Pain is a major symptom of many medical conditions and can significantly reduce a person?s quality of life. The most effective analgesics for pain management are drugs that selectively target the brain?s mu-opioid receptors (MOR) and trigger downstream neuronal responses to suppress pain. However, repeated use of opioid painkillers desensitizes the MORs and causes the development of tolerance, which results in less effective pain relief and prompts an increase in the dose needed over time. Opioid tolerance may contribute to misuse and/or abuse of opioid agonist drugs and may ultimately lead to the problem of opioid addiction and overdose death. The goal of this project is to develop and validate PET/MRI techniques for measuring MOR desensitization and to investigate the molecular and functional mechanisms of opioid tolerance in vivo. Compelling in vitro evidence has demonstrated that desensitized MORs have an increased affinity for ligand binding, which can potentially be detected by positron emission tomography (PET). However, MOR agonists cause concurrent changes in receptor occupancy (due to drug-radiotracer competition) and receptor affinity (due to MOR desensitization). Differentiating the two competing mechanisms with PET becomes difficult, and quantifying receptor occupancy using the classic occupancy model is subject to errors. As MOR desensitization attenuates (if not completely terminates) downstream signaling, a reduction in agonist-induced fMRI responses is expected when more MORs are desensitized. Therefore, simultaneous measurement of MOR agonist-induced PET and fMRI signal changes may offer a new way to dissociate the two competing molecular mechanisms and provides an in vivo estimation of MOR desensitization. We will use a simultaneous PET/magnetic resonance (MR) scanner allowing concurrent and dynamic measurement of PET and fMRI responses. In addition, we have recently developed a novel framework to relate drug-induced hemodynamic response to receptor occupancy. In the study, we will refine the PET/MRI model by incorporating changes in receptor affinity due to receptor trafficking to attribute PET signal changes to specific processes. Our model will be informed by novel studies using desensitization-deficient mice model (?-arrestin knock-out mice) and biased MOR agonists that selectively activate a specific molecular pathway leading to analgesic efficacy (G-protein biased) or another that leads to aversion (?-arrestin biased) in translational relevant nonhuman primates. When successful, the proposed work will enable innovative neuroimaging tools for assess opioid tolerance in patients and help optimize dose escalation strategies, which may help address critical issues in chronic pain management and is timely to combat the current opioid epidemic in the US.